Figure 2.

Ectopic PLAG1 and USF2 Bind the MSI2 Promoter and Enhance Its Transcription in K562 Cells

(A) DNA sequence of the minimal promoter for MSI2 with predicted (MatInspector) USF2 (blue) and PLAG1 (red) binding sites shown. The translation start site of MSI2 is identified by the forward arrow from lower case nucleotides and the initiation codon is underlined.

(B) ChIP-qPCR for PLAG1 and USF2 binding of the minimal MSI2 promoter (n = 4 experiments).

(C) USF2-overexpressing lentiviral construct schematic (top) and western blot quantification of its enhanced protein levels in K562 cells (bottom).

(D) Wild-type (WT) MSI2 minimal promoter reporter construct luciferase activity upon USF2 overexpression (n = 3 experiments).

(E) Luciferase activity of USF2 binding site-mutated (MUT) reporter with and without USF2 overexpression compared with WT (n = 4 experiments).

(F) Endogenous MSI2 transcript levels following USF2 overexpression in K562 cells (n = 3 experiments).

(G) Characterization of PLAG1 isoform expression by western blot in K562 cells.

(H) Comparison of PLAG1-A and PLAG1-B expression in flow-sorted CB HSPCs (performed once from pooled CB samples).

(I) FLAG-tagged PLAG1 short-isoform overexpressing lentiviral construct schematic and western blot quantification of enhanced protein levels in K562 cells (bottom).

(J) WT reporter construct luciferase activity upon PLAG1-B or PLAG1-S overexpression (n = 4 experiments).

(K) Luciferase activity of PLAG1-B and PLAG1-S MUT reporters with PLAG1-B or PLAG1-S overexpression compared with WT (n = 4 experiments).

(L) Endogenous MSI2 transcript levels following PLAG1-B or PLAG1-S overexpression in K562 cells (n = 3 experiments).

Data are presented as means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S1 and Table S3.